ISOCITRATE LYASE FROM CEPHALOSPORIUM-ACREMONIUM - ROLE OF MG2-SITE OFTHE ENZYME( IONS, KINETICS, AND EVIDENCE FOR A HISTIDINE RESIDUE IN THE ACTIVE)

Isocitrate lyase was purified from Cephalosporium acremonium CW-19 fro m cultures growing with poly(oxyethylene)sorbitan monopalmitate as the carbon source. Its subunit M(r) and native M(r) were 63 000 +/- 2000 and 250 000 +/- 5000, respectively. We found the Mg2+-isocitrate compl ex to be the true substrate and that Mg2+ ions act as a nonessential a ctivator, according to the model reported by Giachetti et al. (1988) [ Giachetti, E., Pinzauti, G., Bonaccorsi, R., and Vanni, P. (1988) fur. J. Biochem. 172, 85-91], from which the kinetic parameters were calcu lated. The kinetic study is consistent with an ordered Uni-Bi mechanis m, and the kinetic and rate constants of the model were calculated. pH dependence of the cleavage reaction indicated that the catalysis was dependent on two dissociable groups on the enzyme-substrate complex. T he enzyme was inactivated by diethyl pyrocarbonate following first-ord er kinetics at all reagent concentrations used. The pseudo-first-order rate constant of inactivation increases with pH, suggesting participa tion of an amino acid residue with pK 6.0. Hydroxylamine added to the inactivated enzyme quickly restored the incremental absorption at 240 nm and most of the activity. Data analyses indicated that diethyl pyro carbonate inactivation is a consequence of modification of 11 histidin e residues per enzyme subunit, and from statistical analysis, we concl uded that one is catalytically important. Mg2+-isocitrate protects the enzyme against diethyl pyrocarbonate inactivation with a K-s value of 26.8 +/- 2.1 mu M, close to the K-m value. Isocitrate protects the en zyme but a high concentration, suggesting its binding to the catalytic site of the nonactivated enzyme. Mg2+ ions also produced total compet itive protection.